Method of estimating the amount of a methylated locus in a sample

ABSTRACT

A method of estimating the amount of a methylated locus is provided. In certain embodiments the method comprises: digesting a nucleic acid sample that contains both unmethylated and methylated copies of a genomic locus with an MspJI family member to produce a population of fragments that are in the range of 20-40 nucleotides in length, ligating adaptor sequence A and adaptor sequence B to the respective ends of a target fragment of sequence X, and quantifying the amount of ligation products of formula A-X-B. A kit for performing the method is also provided.

CROSS-REFERENCING

This application claims the benefit of U.S. provisional patentapplication Ser. No. 62/037,057, filed on Aug. 13, 2014, whichapplication is incorporated by reference for all purposes.

BACKGROUND

5-Methylcytosine is a methylated form of the DNA base cytosine that isbelieved to be involved in transcriptional regulation. When cytosine ismethylated, the DNA maintains the same sequence, but the expression ofmethylated genes can be altered.

The function of this chemical varies significantly among species: inbacteria, 5-methylcytosine can be found at a variety of sites, and isoften used as a marker to protect DNA from being cut by nativemethylation-sensitive restriction enzymes; in plants, 5-methylcytosineoccurs at CpG, CpHpG and CpHpH sequences (where H=A, C or T); and, infungi and animals, 5-methylcytosine predominantly occurs at CpGdinucleotides. Most eukaryotes methylate only a small percentage ofthese sites, but 70-80% of CpG cytosines are methylated in vertebrates.

Cytosine methylation in vertebrates typically occurs at CpG sites(cytosine-phosphate-guanine sites, that is, where a cytosine is directlyfollowed by a guanine in the DNA sequence). The formation of Me-CpG iscatalyzed by the enzyme DNA methyltransferase. About 80-90% of CpG sitesare methylated in human DNA, but there are certain areas, known as CpGislands, wherein none of the CpG dinucleotides are methylated. These areassociated with the promoters of 56% of mammalian genes, including allubiquitously expressed genes. One to two percent of the human genome areCpG clusters, and there is an inverse relationship between CpGmethylation and transcriptional activity.

The method described herein provides a way to estimate the amount of amethylated locus in a sample.

SUMMARY

A method of estimating the amount of a methylated locus in a sample isprovided. In certain embodiments, the method may comprise: (a) digestinga nucleic acid sample that contains both unmethylated and methylatedcopies of a genomic locus with an MspJI family member to produce apopulation of fragments that are in the range of 20-40 base pairs inlength and have a central methylated cytosine; (b) ligating adaptorsequence A and adaptor sequence B to the respective ends of a targetfragment of sequence X by: (i) hybridizing a splint oligonucleotide offormula B′-X′-A′ to the fragments of (a) in the presence of the adaptorsequences A and B, wherein X′, A′ and B′ are complementary to X, A andB, respectively, and (ii) ligating adaptor sequence A, sequence X andadaptor sequence B to one another to produce a product of formula A-X-B;and (c) quantifying the amount of ligation products of formula A-X-B,thereby providing an estimate of the amount of the methylated locus inthe nucleic acid sample.

The quantifying may be done using any convenient method, including byqPCR or by sequencing. In some embodiments, the adaptor sequences A andB may be present in the same oligonucleotide molecule and the product ofstep (b) is a circular nucleic acid molecule. In these embodiments, thequantifying may be done by (i) amplifing the circular nucleic acidmolecule by rolling circle amplification (RCA), (ii) hybridizing the RCAproduct to a population of labeled oligonucleotides that hybridize tomultiple positions in the RCA product; and (iii) individually countingthe number of labeled RCA complexes.

As will be discussed in greater detail below, in certain cases themethod may be used to estimate the amount of fetal DNA in a sample ofcell-free DNA from a pregnant female.

A kit for performing the method is also provided.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 schematically illustrates some of the features of an embodimentof the subject method.

FIG. 2 schematically illustrates some of the features of oneimplementation of the subject method.

FIG. 3 schematically illustrates some of the features of anotherimplementation of the subject method.

FIG. 4 schematically illustrates a method by which ligation products canbe counted.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a primer”refers to one or more primers, i.e., a single primer and multipleprimers. It is further noted that the claims can be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

As used herein, the term “genomic locus” refers to a defined region in agenome. A genomic locus exists at the same location in the genomes ofdifferent cells from the same individual, or in different individuals. Agenomic locus in one cell or individual has a nucleotide sequence thatis identical or very similar (i.e., more than 99% identical) to the samegenomic locus in a different cell or individual. The difference innucleotide sequence between the same locus in different cells orindividuals may be due to one or more nucleotide substitutions. Agenomic locus may be defined by genomic coordinates, by name, or using asymbol.

As used herein, the term “methylation state” refers to the presence orabsence of a methyl group on a cytosine residue at a site ofmethylation. For clarity, a cytosine that is unmethylated will bereferred to as “unmethylated cytosine” or “unmethylated C”, and acytosine that is methylated (i.e., 5-methylcytosine) will be referred toas “methylated cytosine”, methylated “C” or “methyl C”.

As used herein, a “site of methylation” refers to the position of acytosine nucleotide that is known to be at least sometimes methylated ina genomic locus. The cytosine at a site of methylation can be anunmethylated cytosine or a methylated cytosine. In other words, the term“site of methylation” refers to a specific cytosine in a genomic locusthat can be in a methylated state. The site of methylation may bedefined by genomic coordinates, or coordinates relative to the startcodon of a gene, for example.

The term “corresponds to” and grammatical equivalents, e.g.,“corresponding”, as used herein refers to a specific relationshipbetween the elements to which the term refers. For example, anoligonucleotide that corresponds to a sequence in a longer nucleic acidcontains the same nucleotide sequence as or is complementary to anucleotide sequence in the nucleic acid.

In the context of a nucleotide in an oligonucleotide that corresponds toa site of methylation or a nucleotide in an oligonucleotide thatcorresponds to a methylated cytosine, the term “corresponds to” andgrammatical equivalents thereof are intended to identify the nucleotidethat is correspondingly positioned relative to (i.e., positioned acrossfrom) a site of methylation when the two nucleic acids (e.g., anoligonucleotide and genomic DNA containing a methylated cytosine) arealigned or base paired. Again, unless otherwise indicated (e.g., in thecase of a nucleotide that “does not base pair” or “base pairs” with aparticular residue) a nucleotide that “corresponds to” a site ofmethylation base pairs with either a methylated site or an unmethylatedsite. For clarity, in an oligonucleotide, a G or C nucleotide at aposition that corresponds to a methylated cytosine in a sequence, e.g.,a genomic locus, can: a) base pair with a methylated cytosine in thesequence, b) base pair with a cytosine that positionally corresponds tothe methylated cytosine in an amplified version of the sequence, or c)base pair with a G residue that is complementary to such a cytosine inan amplified sequence.

As used herein, a “sequence that is methylated” is a nucleotide sequencethat contains a site of methylation, i.e., a cytosine nucleotide that isknown to be at least sometimes methylated.

As used herein, the term “unmethylated”, with reference to a nucleotidesequence, refers to the copies of a sequence that are not methylated.

As used herein, the term “methylated”, with reference to a nucleotidesequence, refers to copies of a sequence that contain 5-methylcytosine.Methylation of a genomic locus may, e.g., alter the expression of aprotein, which causes a phenotypic change (e.g., a cancer-relatedphenotype) in the cells that have such a methylated locus.Alternatively, methylation of a genomic locus may be silent.

A sample that comprises “both unmethylated and methylated copies of agenomic locus” and grammatical equivalents thereof, refers to a samplethat contains multiple DNA molecules of the same genomic locus, wherethe sample contains both unmethylated copies of the genomic locus andmethylated copies of the same locus. In this context, the term “copies”is not intended to mean that the sequences were copied from one another.Rather, the term “copies” is intended to indicate that the sequences areof the same locus in different cells or individuals. In other words, asample contains a mixture of nucleic acid molecules having the samenucleotide sequence, except that some of the molecules containmethylated cytosine residues.

As used herein, the term “degree of methylation” refers to the relativenumber, percentage, or fraction of members of a particular targetnucleotide species within a sample that are methylated compared to thosemembers of that particular target nucleotide species that are notmethylated.

The term “mixture”, as used herein, refers to a combination of elements,that are interspersed and not in any particular order. A mixture isheterogeneous and not spatially separable into its differentconstituents. Examples of mixtures of elements include a number ofdifferent elements that are dissolved in the same aqueous solution and anumber of different elements attached to a solid support at randompositions (i.e., in no particular order). A mixture is not addressable.To illustrate by example, an array of spatially separated surface-boundpolynucleotides, as is commonly known in the art, is not a mixture ofsurface-bound polynucleotides because the species of surface-boundpolynucleotides are spatially distinct and the array is addressable.

The term “nucleotide” is intended to include those moieties that containnot only the known purine and pyrimidine bases, but also otherheterocyclic bases that have been modified. Such modifications includemethylated purines or pyrimidines, acylated purines or pyrimidines,alkylated riboses or other heterocycles. In addition, the term“nucleotide” includes those moieties that contain hapten or fluorescentlabels and may contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, are functionalized as ethers, amines, or the likes.

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length, e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, up to about10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotidesor ribonucleotides, and may be produced enzymatically or synthetically(e.g., PNA as described in U.S. Pat. No. 5,948,902 and the referencescited therein) which can hybridize with naturally occurring nucleicacids in a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in Watson-Crick basepairing interactions. Naturally-occurring nucleotides include guanine,cytosine, adenine, thymine, uracil (G, C, A, T and U respectively). DNAand RNA have a deoxyribose and ribose sugar backbone, respectively,whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycineunits linked by peptide bonds. In PNA various purine and pyrimidinebases are linked to the backbone by methylene carbonyl bonds. A lockednucleic acid (LNA), often referred to as inaccessible RNA, is a modifiedRNA nucleotide. The ribose moiety of an LNA nucleotide is modified withan extra bridge connecting the 2′ oxygen and 4′ carbon. The bridge“locks” the ribose in the 3′-endo (North) conformation, which is oftenfound in the A-form duplexes. LNA nucleotides can be mixed with DNA orRNA residues in the oligonucleotide whenever desired. The term“unstructured nucleic acid”, or “UNA”, is a nucleic acid containingnon-natural nucleotides that bind to each other with reduced stability.For example, an unstructured nucleic acid may contain a G′ residue and aC′ residue, where these residues correspond to non-naturally occurringforms, i.e., analogs, of G and C that base pair with each other withreduced stability, but retain an ability to base pair with naturallyoccurring C and G residues, respectively. Unstructured nucleic acid isdescribed in US20050233340, which is incorporated by reference hereinfor disclosure of UNA.

The term “target polynucleotide,” as used herein, refers to apolynucleotide of interest under study. In certain embodiments, a targetpolynucleotide contains one or more sequences that are of interest andunder study.

The term “oligonucleotide” as used herein denotes a single-strandedmultimer of nucleotides of from about 2 to 200 nucleotides, up to 500nucleotides in length. Oligonucleotides may be synthetic or may be madeenzymatically, and, in some embodiments, are 30 to 150 nucleotides inlength. Oligonucleotides may contain ribonucleotide monomers (i.e., maybe oligoribonucleotides) or deoxyribonucleotide monomers. Anoligonucleotide may be 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60,61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200 nucleotides inlength, for example.

The term “primer” as used herein refers to an oligonucleotide that iscapable of acting as a point of initiation of synthesis when placedunder conditions in which synthesis of a primer extension product, whichis complementary to a nucleic acid strand, is induced, i.e., in thepresence of nucleotides and an inducing agent such as a DNA polymeraseand at a suitable temperature and pH. The primer may be single-strandedand must be sufficiently long to prime the synthesis of the desiredextension product in the presence of the inducing agent. The exactlength of the primer will depend upon many factors, includingtemperature, source of primer and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide primer typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides. The primersherein are selected to be substantially complementary to differentstrands of a particular target DNA sequence. This means that the primersmust be sufficiently complementary to hybridize with their respectivestrands. Therefore, the primer sequence need not reflect the exactsequence of the template. For example, a non-complementary nucleotidefragment may be attached to the 5′ end of the primer, with the remainderof the primer sequence being complementary to the strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprimer, provided that the primer sequence has sufficient complementaritywith the sequence of the strand to hybridize therewith and thereby formthe template for the synthesis of the extension product.

The term “hybridization” or “hybridizes” refers to a process in which anucleic acid strand anneals to and forms a stable duplex, either ahomoduplex or a heteroduplex, under normal hybridization conditions witha second complementary nucleic acid strand, and does not form a stableduplex with unrelated nucleic acid molecules under the same normalhybridization conditions. The formation of a duplex is accomplished byannealing two complementary nucleic acid strands in a hybridizationreaction. The hybridization reaction can be made to be highly specificby adjustment of the hybridization conditions (often referred to ashybridization stringency) under which the hybridization reaction takesplace, such that hybridization between two nucleic acid strands will notform a stable duplex, e.g., a duplex that retains a region ofdouble-strandedness under normal stringency conditions, unless the twonucleic acid strands contain a certain number of nucleotides in specificsequences which are substantially or completely complementary. “Normalhybridization or normal stringency conditions” are readily determinedfor any given hybridization reaction. See, for example, Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork, or Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press. As used herein, the term “hybridizing”or “hybridization” refers to any process by which a strand of nucleicacid binds with a complementary strand through base pairing.

A nucleic acid is considered to be “selectively hybridizable” to areference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Moderate and high stringency hybridizationconditions are known (see, e.g., Ausubel, et al., Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold SpringHarbor, N.Y.). One example of high stringency conditions includeshybridization at about 42 C in 50% formamide, 5×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 ug/ml denatured carrier DNA followed bywashing two times in 2×SSC and 0.5% SDS at room temperature and twoadditional times in 0.1×SSC and 0.5% SDS at 42° C.

The term “duplex,” or “duplexed,” as used herein, describes twocomplementary polynucleotides that are base-paired, i.e., hybridizedtogether.

The term “amplifying” as used herein refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid. Amplifying a nucleic acidmolecule typically includes denaturing the template nucleic acid,annealing primers to the template nucleic acid at a temperature that isbelow the melting temperatures of the primers, and enzymaticallyelongating from the primers to generate an amplification product. Thedenaturing, annealing and elongating steps each can be performed once.Generally, however, the denaturing, annealing and elongating steps areperformed multiple times such that the amount of amplification productis increasing, often times exponentially, although exponentialamplification is not required by the present methods. Amplificationtypically requires the presence of deoxyribonucleoside triphosphates, aDNA polymerase enzyme and an appropriate buffer and/or co-factors foroptimal activity of the polymerase enzyme. The term “amplificationproduct” refers to the nucleic acid sequences, which are produced fromthe amplifying process as defined herein. An amplification reaction canbe isothermal (e.g., in the case of rolling circle amplification) or mayrequire thermocycling (in the case of PCR).

The terms “determining,” “measuring,” “evaluating,” “assessing,”“assaying,” and “analyzing” are used interchangeably herein to refer toany form of measurement, and include determining if an element ispresent or not. These terms include both quantitative and/or qualitativedeterminations. Assessing may be relative or absolute. “Assessing thepresence of” includes determining the amount of something present, aswell as determining whether it is present or absent.

The term “partitioning”, with respect to a genome, refers to theseparation of one part of the genome from the remainder of the genome toproduce a product that is isolated from the remainder of the genome. Theterm “partitioning” encompasses enriching.

The term “genomic region”, as used herein, refers to a region of agenome, e.g., an animal or plant genome such as the genome of a human,monkey, rat, fish or insect or plant. In certain cases, anoligonucleotide used in the method described herein may be designedusing a reference genomic region, i.e., a genomic region of knownnucleotide sequence, e.g., a chromosomal region whose sequence isdeposited at NCBI's Genbank database or other databases, for example.Such an oligonucleotide may be employed in an assay that uses a samplecontaining a test genome, where the test genome contains a binding sitefor the oligonucleotide.

The term “genomic sequence”, as used herein, refers to a sequence thatoccurs in a genome.

The term “genomic fragment”, as used herein, refers to a region of agenome, e.g., an animal or plant genome such as the genome of a human,monkey, rat, fish or insect or plant. A genomic fragment may be anentire chromosome, or a fragment of a chromosome.

The term “affinity tag”, as used herein, refers to moiety that can beused to separate a molecule to which the affinity tag is attached fromother molecules that do not contain the affinity tag. An “affinity tag”is a member of a specific binding pair, i.e. two molecules where one ofthe molecules through chemical or physical means specifically binds tothe other molecule. The complementary member of the specific bindingpair, referred to herein as a “capture agent” may be immobilized (e.g.,to a chromatography support, a bead or a planar surface) to produce anaffinity chromatography support that specifically binds the affinitytag. In other words, an “affinity tag” may bind to a “capture agent”,where the affinity tag specifically binds to the capture agent, therebyfacilitating the separation of the molecule to which the affinity tag isattached from other molecules that do not contain the affinity tag.

As used herein, the term “biotin moiety” refers to an affinity agentthat includes biotin or a biotin analogue such as desthiobiotin,oxybiotin, 2′-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin,etc. Biotin moieties bind to streptavidin with an affinity of at least10⁻⁸M. A biotin affinity agent may also include a linker, e.g.,-LC-biotin, -LC-LC-Biotin, -SLC-Biotin or -PEG.-Biotin where n is 3-12.

The term “terminal nucleotide”, as used herein, refers to the nucleotideat either the 5′ or the 3′ end of a nucleic acid molecule. The nucleicacid molecule may be in double-stranded form (i.e., duplexed) or insingle-stranded form.

The term “ligating”, as used herein, refers to the enzymaticallycatalyzed joining of the terminal nucleotide at the 5′ end of a firstDNA molecule to the terminal nucleotide at the 3′ end of a second DNAmolecule.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have at least 10, at least 100, at least 100, at least 10,000, atleast 100,000, at least 10⁶, at least 10⁷, at least 10⁸ or at least 10⁹or more members.

If two nucleic acids are “complementary”, they hybridize with oneanother under high stringency conditions. The term “perfectlycomplementary” is used to describe a duplex in which each base of one ofthe nucleic acids base pairs with a complementary nucleotide in theother nucleic acid. In many cases, two sequences that are complementaryhave at least 10, e.g., at least 12 or 15 nucleotides of complementarityand in certain cases may have one, two or three non-complementary bases.

The term “digesting” is intended to indicate a process by which anucleic acid is cleaved by a restriction enzyme. In order to digest anucleic acid, a restriction enzyme and a nucleic acid containing arecognition site for the restriction enzyme are contacted underconditions suitable for the restriction enzyme to work. Conditionssuitable for activity of commercially available restriction enzymes areknown, and supplied with those enzymes upon purchase.

An “oligonucleotide binding site” refers to a site to which anoligonucleotide hybridizes in a target polynucleotide. If anoligonucleotide “provides” a binding site for a primer, then the primermay hybridize to that oligonucleotide or its complement.

The term “separating”, as used herein, refers to physical separation oftwo elements (e.g., by size or affinity, etc.) as well as degradation ofone element, leaving the other intact.

The term “reference chromosomal region,” as used herein refers to achromosomal region of known nucleotide sequence, e.g. a chromosomalregion whose sequence is deposited at NCBI's Genbank database or otherdatabases, for example.

The term “strand” as used herein refers to a nucleic acid made up ofnucleotides covalently linked together by covalent bonds, e.g.,phosphodiester bonds.

In a cell, DNA usually exists in a double-stranded form, and as such,has two complementary strands of nucleic acid referred to herein as the“top” and “bottom” strands. In certain cases, complementary strands of achromosomal region may be referred to as “plus” and “minus” strands, the“first” and “second” strands, the “coding” and “noncoding” strands, the“Watson” and “Crick” strands or the “sense” and “antisense” strands. Theassignment of a strand as being a top or bottom strand is arbitrary anddoes not imply any particular orientation, function or structure. Thenucleotide sequences of the first strand of several exemplary mammalianchromosomal regions (e.g., BACs, assemblies, chromosomes, etc.) isknown, and may be found in NCBI's Genbank database, for example.

The term “top strand,” as used herein, refers to either strand of anucleic acid but not both strands of a nucleic acid. When anoligonucleotide or a primer binds or anneals “only to a top strand,” itbinds to only one strand but not the other. The term “bottom strand,” asused herein, refers to the strand that is complementary to the “topstrand.” When an oligonucleotide binds or anneals “only to one strand,”it binds to only one strand, e.g., the first or second strand, but notthe other strand.

The term “covalently linking” refers to the production of a covalentlinkage between two separate molecules, e.g., the top and bottom strandsof a double stranded nucleic acid. Ligating is a type of covalentlinking.

The term “denaturing,” as used herein, refers to the separation of atleast a portion of the base pairs of a nucleic acid duplex by placingthe duplex in suitable denaturing conditions.

Denaturing conditions are well known in the art. In one embodiment, inorder to denature a nucleic acid duplex, the duplex may be exposed to atemperature that is above the melting temperature of the duplex, therebyreleasing one strand of the duplex from the other. In certainembodiments, a nucleic acid may be denatured by exposing it to atemperature of at least 90° C. for a suitable amount of time (e.g., atleast 30 seconds, up to 30 mins). Nucleic acids may also be denaturedchemically (e.g., using urea or NaOH).

As used herein, the term “label” refers to any atom or molecule that canbe used to provide a detectable (preferably quantifiable) effect, andthat can be attached to a nucleic acid or protein. Labels include butare not limited to dyes and radiolabels such as ³²P; binding moietiessuch as biotin; haptens such as digoxigenin; luminogenic, phosphorescentor fluorogenic moieties; and fluorescent dyes alone or in combinationwith moieties that can suppress or shift emission spectra byfluorescence resonance energy transfer (FRET). Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like. A label may be a charged moiety (positive ornegative charge) or alternatively, may be charge neutral. Labels caninclude or consist of a nucleic acid or a protein sequence, so long asthe sequence comprising the label is detectable.

The term “labeled oligonucleotide”, as used herein, refers to anoligonucleotide that has an affinity tag (e.g., a biotin moiety), anoligonucleotide modified with atoms or groups enabling separation ordetection (e.g., bromo-deoxyuridine, or colloidal gold particlesconferring different density), and an oligonucleotide modified with oran optically detectable label (e.g., a fluorescence or another type oflight emitting label). Oligonucleotides that contain only naturallyoccurring nucleotides are not labeled oligonucleotides.

The term “sequencing”, as used herein, refers to a method by which theidentity of at least 10 consecutive nucleotides (e.g., the identity ofat least 20, at least 50, at least 100 or at least 200 or moreconsecutive nucleotides) of a polynucleotide is obtained.

The term “next-generation sequencing” refers to the so-calledparallelized sequencing-by-synthesis or sequencing-by-ligation platformscurrently employed by Illumina, Life Technologies, and Roche, etc.Next-generation sequencing methods may also include nanopore sequencingmethods or electronic-detection based methods such as Ion Torrenttechnology commercialized by Life Technologies.

The term “extending”, as used herein, refers to the extension of aprimer by the addition of nucleotides using a polymerase. If a primerthat is annealed to a nucleic acid is extended, the nucleic acid acts asa template for an extension reaction.

The term “clonal PCR” is a PCR technique in which each reaction is doneon a single template molecule, and the PCR reactions are kept spatiallyseparated from one another. Bridge PCR and emulsion PCR, commonly usedin next generation sequencing applications, are examples of clonal PCR.

The term “bridge PCR” refers to a solid-phase polymerase chain reactionin which the primers that are extended in the reaction are tethered to asubstrate by their 5′ ends. During amplification, the amplicons form abridge between the tethered primers. Bridge PCR (which may also bereferred to as “cluster PCR”) is used in Illumina's Solexa platform.Bridge PCR and Illumina's Solexa platform are generally described in avariety of publications, e.g., Gudmundsson et al (Nat. Genet. 200941:1122-6), Out et al (Hum. Mutat. 2009 30:1703-12) and Turner (Nat.Methods 2009 6:315-6), U.S. Pat. No. 7,115,400, and US applicationpublication Nos. US20080160580 and US20080286795. Bridge PCR is a typeof “clonal PCR”, i.e., is a PCR technique in which each reaction isbegun on a single template molecule, and the PCR reactions are keptspatially separated from one another.

The term “barcode sequence” or “molecular barcode”, as used herein,refers to a unique sequence of nucleotides used to a) identify and/ortrack the source of a polynucleotide in a reaction and/or b) count howmany times an initial molecule is sequenced (e.g., in cases wheresubstantially every molecule in a sample is tagged with a differentsequence, and then the sample is amplified). A barcode sequence may beat the 5′-end, the 3′-end or in the middle of an oligonucleotide.Barcode sequences may vary widely in size and composition; the followingreferences provide guidance for selecting sets of barcode sequencesappropriate for particular embodiments: Brenner, U.S. Pat. No.5,635,400; Brenner et al, Proc. Natl. Acad. Sci., 97: 1665-1670 (2000);Shoemaker et al, Nature Genetics, 14: 450-456 (1996); Morris et al,European patent publication 0799897A1; Wallace, U.S. Pat. No. 5,981,179;and the like. In particular embodiments, a barcode sequence may have alength in range of from 4 to 36 nucleotides, or from 6 to 30nucleotides, or from 8 to 20 nucleotides.

As used herein, the term “PCR reagents” refers to all reagents that arerequired for performing a polymerase chain reaction (PCR) on a template.As is known in the art, PCR reagents essentially include a first primer,a second primer, a thermostable polymerase, and nucleotides. Dependingon the polymerase used, ions (e.g., Mg²⁺) may also be present. PCRreagents may optionally contain a template from which a target sequencecan be amplified.

The term, “intramolecularly ligating” refers to a ligation in which the5′ end and the 3′ end of a strand of nucleic acid are ligated to oneanother to produce a circular DNA molecule. As used herein, the term“MspJI family restriction endonuclease” refers to a family ofrestriction endonucleases that recognize methylated cytosine andgenerate a double stranded break in the DNA molecule at a site that isupstream or downstream from the methylated cytosine. These enzymes donot cleave unmethylated DNA. If both strands of the DNA are methylated(which is commonly the case in CpG methylation) then the enzyme will cutthe DNA to produce a fragment of 20-40 base pairs in length, dependingon the enzyme used. MspJI, for example, recognizes each hemi-methylatedsite individually and cleaves bidirectionally to generate 32-base or31-base fragments, respectively. These fragments contain the centralmethylated site and have 4-base 5′ overhangs at each end. The MspJIfamily of endonucleases is described in, e.g., Zheng et al (NucleicAcids Res. 2010 A unique family of Mrr-like modification-dependentrestriction endonucleases. 38: 5527-34) and Cohen-Karni (Proc. Natl.Acad. Sci. 2011 The MspJI family of modification-dependent restrictionendonucleases for epigenetic studies 108:11040-5) and US20100167942,which are incorporated by reference for details of this family ofenzymes. Examples of MspJI family restriction endonucleasse includeFspEI, LpnPI, AspBHI, RlaI, and SgrTI. Reference to a MspJI familymember, either generically or by name, is intended to refer to a wildtype restriction endonuclease as well as variants that have an aminoacid sequence that is at least 90% (e.g., at least 95%) identical to thewild type restriction endonuclease.

As used herein, the term “population of fragments that are in the rangeof 20-40 base pairs in length” refers to a mixture of digestionfragments. The fragments have a central cytosine residue (which is partof the recognition site for the MspJI family endonuclease) and have 3′or 5′ overhangs, depending on which enzyme is used.

As used herein, the term “respective ends”, in the phrase “ligatingadaptor sequence A and adaptor sequence B to the respective ends of atarget fragment” is intended to mean that sequence A is added to one endof the target fragment and sequence B is added to the other end of thetarget fragment.

Certain polynucleotides described herein may be referred by a formula(e.g., “B′-X′-A′” and “A-X-B”). Such formulas follow the establishedconvention in that they describe a polynucleotide that is oriented inthe 5′ to 3′ direction. The components of the formula, e.g., “A”, “X”and “B” refer to separately definable sequences of nucleotides within apolynucleotide, where the sequences are linked together covalently suchthat a polynucleotide described by a formula is a single molecule. Inmany cases the components of the formula are immediately adjacent to oneanother in the single molecule. Following convention, the complement ofa sequence shown in a formula will be indicated with a prime (′) suchthat the complement of sequence “A” will be “A′”. Moreover, unlessotherwise indicated or implicit from the context, a polynucleotidedefined by a formula may have additional sequence, a primer bindingsite, a molecular barcode, a promoter, or a spacer, etc., at its 3′ end,its 5′ end or both the 3′ and 5′ ends.

As used herein, the term “adaptor sequences A and B” refers to differentsequences.

As used herein, the term “ligatably adjacent” in the context of twooligonucleotide sequences that are ligatably adjacent to one another,means that there are no intervening nucleotides between twooligonucleotides and they can be ligated to one another.

As used herein, the term “splint oligonucleotide”, as used herein,refers to an oligonucleotide that, when hybridized to two or more otherpolynucleotides, acts as a “splint” to position the polynucleotides nextto one another so that they can be ligated together, as illustrated inFIG. 1.

As used herein, the term “a circular nucleic acid molecule” refers to astrand that is in the form of a closed circle that has no free 3′ or 5′ends.

As used herein, the term “fetal fraction” refers to the percentage ofcell free DNA from a developing fetus in the maternal bloodstream of apregnant female.

Other definitions of terms may appear throughout the specification.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

A method of estimating the amount of a methylated locus is provided. Incertain embodiments the method comprises: digesting a nucleic acidsample that contains both unmethylated and methylated copies of agenomic locus with an MspJI family member to produce a population offragments that are in the range of 20-40 nucleotides in length, ligatingadaptor sequence A and adaptor sequence B to the respective ends of atarget fragment of sequence X, and quantifying the amount of ligationproducts of formula A-X-B. A kit for performing the method is alsoprovided.

Before the various embodiments are described, it is to be understoodthat the teachings of this disclosure are not limited to the particularembodiments described, and as such can, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present teachings will be limited onlyby the appended claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, the someexemplary methods and materials are now described.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can need to be independentlyconfirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

With reference to FIG. 1, some embodiments of the method comprisedigesting a nucleic acid sample 2 that contains both unmethylated (4 and6) and methylated copies (8) of a genomic locus using an MspJI familyrestriction endonuclease. As illustrated, if the recognition sequence ismethylated on both strands (indicated by the filled circle 10), then theenzyme will cleave on both sides of the recognition sequence, asindicated by the x's in fragment 8. This digest produces digested sample12 that comprises a population of fragments that are in the range of20-40 base pairs in length and have a central methylated cytosine. Aswould be apparent, the initial sample being digested is not an amplifiedsample. If genomic DNA is digested, the digested sample should containat least several thousand, if not at least at least 10,000 or 100,000 ormore, fragments that are 20-40 base pairs in length and correspond todifferent methylated loci, as well as longer fragments of the same locithat have not been digested into a small fragment because they are notmethylated. One of these fragments that are in the range of 20-40 basepairs in length corresponds to target fragment 14 of sequence X. In thismethod, target fragment 14 is selected by ligating adaptor sequence A 16and adaptor sequence B 18 to its ends. This is done by (i) hybridizing asplint oligonucleotide 20 of formula B′-X′-A′ to the fragments of 12 inthe presence of the adaptor sequences A 16 and B 18, wherein X′, A′ andB′ are complementary to X, A and B, respectively, to make duplex 21 and(ii) ligating adaptor sequence A 16, the fragment of sequence X 14 andadaptor sequence B 18 to one another to produce a product 22 of formulaA-X-B. As would be apparent, splint oligonucleotide 20 and adaptorsequences A 16 and B 18 are designed so that the ends of sequences A 16and B 18 are ligatably adjacent to the ends of the target fragment ofsequence X when they are hybridized to splint oligonucleotide 20 induplex 21. Splint oligonucleotide 20 and adaptor sequences A 16 and B 18are shown as duplex 19 in FIG. 1 solely to show which sequences arecomplementary to one another. In practice, splint oligonucleotide 20 andadaptor sequences A 16 and B 18 are not usually hybridized with oneanother prior to hybridization with the sample. In practice, the variousoligonucleotides can be combined with the digested sample in a singlevessel, and the mixture heated and then cooled, thereby denaturing andannealing the various sequences to one another prior to ligation. Afterligating adaptor sequence A 16, the fragment of sequence X 14 andadaptor sequence B 12 to one another to produce a product 22 of formulaA-X-B, the amount of product 22 can be quantified, thereby providing anestimate of the amount of the methylated locus in nucleic acid sample 2.In particular embodiments, the ligase used may be a thermostable ligase,and the ligation step may be done by cycling the reaction throughmultiple rounds of denaturation and renaturation, thereby driving thereaction to completion. Quantification of product 22 may be done avariety of different ways, e.g., by quantitative PCR, by sequencing andby digital counting, examples of which are described in greater detailbelow. In certain embodiments, the splint probes and adaptor sequencemay be designed and/or ligated using a method described in UK patentapplication serial no. 1321191.7, filed on Dec. 2, 2013, which patentapplication is incorporated by reference herein for disclosure of thosemethods.

In the embodiment illustrated in FIG. 2, adaptor sequences A 16 and B 18may be in separate oligonucleotide molecules comprisingnon-complementary sequences 40 and 42, that provide primer binding sitesin product 22. In this embodiment, product 22 can be amplified usingprimers 44 and 46. In this embodiment, the amount of product 22 can bequantifying by any suitable qPCR assay, e.g., a TaqMan assay or thelike. In another embodiment, product 22 may be sequenced (with orwithout amplification) using primers 44 and 46, or primers thathybridize to tails that have been added to primers 44 and 46, forexample. In these embodiments, the amount of product 22 can be estimatedby counting the number of sequencereads corresponding to sequence X. Inalternative embodiments, non-complementary sequences 40 and/or 42 maycontain a molecular barcode and each molecule of product 22 contains adifferent barcode sequence. In these embodiments, the amount of product22 can be estimated by counting the number of unique barcode sequencesthat are associated with the sequence reads corresponding to sequence X.

In another embodiment illustrated in FIG. 3, adaptor sequences A and Bare present in a single oligonucleotide molecule 50 and product 22 is acircular nucleic acid molecule. In these embodiments, product 22 may bequantified by amplifying the product by rolling circle amplification(RCA) (e.g., using primer 52, which may be complementary to a sequenceanywhere in the product) and then estimating the number of RCA productsproduced. The rolling circle amplification products will containmultiple copies of each of the sequences in product 22. In theseembodiments, the quantifying may be done by hybridizing the RCA productto a population of labeled oligonucleotides that hybridize to multiplepositions in the RCA product; and individually counting the number oflabeled RCA complexes.

FIG. 4 illustrates one way by which this embodiment may be implemented.In this implementation, product 22 is acomposed of four productmolecules 22 a, 22 b, 22 c and 22 d. In practice, product 22 may becomposed of at least 100, at least 1,000 or at least 10,000 or moreproduct molecules of formula A-X-B. In this embodiment, the products areamplified by rolling circle amplification using primer 52 (which may becomplementary to a sequence anywhere in the product) to produce aplurality of RCA products. The number of rolling circle amplificationproducts can be estimated by distributing the RCA products on thesurface of a support (a slide), hybridizing the RCA products usinglabeled oligonucleotides (e.g., fluorescently labeled oligonucleotides)and then counting the number of discrete signals in an area of thesupport by microscopy, e.g., fluorescence microscopy. The labeling canbe done before or after the products have been distributed on thesupport and, because each RCA product contains thousands of copies ofthe same sequences, there should be thousands of binding sites for thelabeled oligonucleotides, thereby increasing the signal. In certainembodiments, the amplification and/or detection methods may beimplemented using a method described in UK patent application serial no.1321196.6, filed on Dec. 2, 2013, which patent application isincorporated by reference herein for disclosure of those methods.

The amount of the methylated locus in the sample may be expressed in avariety of different ways. For example, in some embodiments, the resultsobtained in the quantification step may be normalized to the amount ofinput DNA and expressed as a measurement per amount of input DNA (e.g.,x number of molecules in y amount of input DNA). This number can providea useful metric when compared to the amount of other sequences in thesame sample. For example, the relative amount (e.g., the percentage) ofthe methylated locus in the sample may be calculated by comparing theresults obtained by the present method to results obtained for areference locus that is known to be always methylated in the sample. Inthis example, the amount of the reference locus in the sample can bequantified using a similar method (where the same enzyme or anotherMspJI family enzyme may be used and the target-specific sequences in thesplint oligonucleotide hybridizes to a fragment corresponding to areference locus, not locus X). In another example, the relative amount(e.g., the percentage) of the methylated locus in the sample may becalculated by comparing the results obtained by the present method toresults obtained for a reference locus that is known to be present inthe sample. In this example, the amount of the reference locus in thesample can be quantified using a similar method (where an enzyme otherthan a MspJI family enzyme, e.g., a methylation insensitive enzyme, maybe used and the target-specific sequences in the splint oligonucleotidehybridizes to a fragment corresponding to the reference locus, not X).In some embodiments, the absolute amount of methylated locus in thesample can be compared to a standard curve (e.g., a standard curvegenerated using control samples that contain known amounts of themethylated locus) to provide an estimate of the absolute number ofmolecules of the methylated locus in the sample.

The lengths of the various regions of the adaptors and splintoligonucleotides may vary greatly depending upon the desired applicationand how much freight (i.e., how many primer binding sites, barcodes,etc.) are carried by the adaptors. In practice, sequence X′ of thesplint oligonucleotide will be designed to match the sequence of afragment that is expected to be generated by digesting a genomic samplewith the MspJI family restriction endonuclease being used in the method.For example, if MspJI is used, then sequence X′ will be about 32nucleotides in length and the ends of X will correspond to the cleavagesites for MspJI in genomic DNA. Other MspJI family members createfragments of different lengths and, as such, the length of X′ does notneed to be about 32 bases if another enzyme, include FspEI, LpnPI,AspBHI, RlaI, and SgrTI, is used. In some cases, X′ may be in the rangeof 25-35 nucleotides in length. Because the recognition sites for theseenzymes are known and the sequence of several genomes, including thehuman genome, are known, the design of splint oligonucleotides can bedone by hand or computationally. Depending on the desired application,adaptor sequences A and B may be of 15 to 100 bases (e.g., 18 to 30bases) in length. As should be readily apparent, the nucleotide sequenceof any additional sequences that are appended to the adaptor sequences,e.g., primer binding sites/barcodes, etc., should be designed so thatthey do not hybridize to the genome under study.

In particular embodiments, one of the adaptor sequences may be linked toan affinity tag, e.g., a biotin moiety, so that, after ligation, theligation products can be separated from unligated molecules using asolid support comprising a surface tethered capture agent for theaffinity tag, thereby binding the ligation products to the solid supportand isolating those molecules from other nucleic acids in the sample.

As would be apparent, the primers used in some embodiments may containsequences that are compatible with use in, e.g., Illumina's reversibleterminator method, Roche's pyrosequencing method (454), LifeTechnologies' sequencing by ligation (the SOLiD platform) or LifeTechnologies' Ion Torrent platform. Examples of such methods aredescribed in the following references: Margulies et al (Nature 2005 437:376-80); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9);Shendure (Science 2005 309: 1728); Imelfort et al (Brief Bioinform. 200910:609-18); Fox et al (Methods Mol Biol. 2009; 553:79-108); Appleby etal (Methods Mol Biol. 2009; 513:19-39) and Morozova (Genomics. 200892:255-64), which are incorporated by reference for the generaldescriptions of the methods and the particular steps of the methods,including all starting products, reagents, and final products for eachof the steps.

In some embodiments, the method may be multiplexed to quantify severalmethylated loci in the sample. This may be done using several splintoligonucleotides, where the sequence corresponding to X is complementaryto the loci under investigation. The method may be used to analyze atleast 2, at least 5, at least 10, at least 50 or at least 100 differentloci in the same reaction.

The method described above can be employed to analyze genomic DNA fromvirtually any organism, including, but not limited to, plants, animals(e.g., reptiles, mammals, insects, worms, fish, etc.), tissue samples,bacteria, fungi (e.g., yeast), phage, viruses, cadaveric tissue,archaeological/ancient samples, etc. In certain embodiments, the genomicDNA used in the method may be derived from a mammal, where in certainembodiments the mammal is a human. In exemplary embodiments, the genomicsample may contain genomic DNA from a mammalian cell, such as, a human,mouse, rat, or monkey cell. The sample may be made from cultured cellsor cells of a clinical sample, e.g., a tissue biopsy, scrape or lavageor cells of a forensic sample (i.e., cells of a sample collected at acrime scene). In particular embodiments, the nucleic acid sample may beobtained from a biological sample such as cells, tissues, bodily fluids,and stool. Bodily fluids of interest include but are not limited to,blood, serum, plasma, saliva, mucous, phlegm, cerebral spinal fluid,pleural fluid, tears, lactal duct fluid, lymph, sputum, cerebrospinalfluid, synovial fluid, urine, amniotic fluid, and semen. In particularembodiments, a sample may be obtained from a subject, e.g., a human. Insome embodiments, the sample analyzed may be a sample of cell-free DNAobtained from blood, e.g., from the blood of a pregnant female.

In certain embodiments, the subject method may be used to determine themethylation status of a target locus that is associated with a disease,e.g., cancer or a liver disease such as chronic hepatitis or cirrhosis,where the methylation status of locus can be used to diagnose thedisease. In these embodiments, source of the genomic DNA may be tissuebiopsy or a bodily fluid that contains DNA derived from diseased tissue.

In certain embodiments, the subject method may be used in non-invasiveprenatal testing (NIPT) for diagnosis of genetic and epigeneticanomalies in the fetus, including fetal aneuploidies such as trisomy 21,trisomy 18 and trisomy 13; imprinting disorders such asBeckwith-Wiedemann syndrome, Prader-Willi and Angelman syndromes, andAlbright's Hereditary Osteodystrophy; and other genetic defects, such asFragile X Syndrome. For instance, hypermethylation of the following locimay be diagnostic: CpG islands around CRYAA, HLCS, C21orf63, OLIG2,CBR1, SIM2, DSCAM, TRPM2, C21orf29, MIR155HG, ICOS ligand-like, SIM2,DSCR6, chr21 group00165, PRMT2 and COL18A1 for trisomy 21 (Lim et al.,BMC Med Genomics, 2014 7:1; Tong et al., Clin Chem, 2009 56:90; Tong etal., PLoS One, 2010 5:e15244; Chim et al., Clin Chem, 2008 54:500; U.S.Pat. No. 8,476,013); hypermethylated CpG sites in the promoter sequenceof SERPINBS (maspin), between the VAPA and APCDD1 genes, and CpG sitesin CIDEA, chr18 group00091, chr18 group00094, KLHL14, ST8SIA3, ONECUT2,RAX, chr18 group00277, NETO1, MBP and NFATC1 for trisomy 18 (Tong etal., Clin Chem, 2006 52:2194; Tsui et al., PLoS One, 2010 5:e15069; U.S.Pat. No. 8,476,013); hypermethylation sites in chr13 group00016, ATP8A2,GSJ1, PDX1, MAB21L1, RB1, PCDH17, KLHL1, POU4F1, GPC6, SOX21, ZIC2,chr13 group00385, chr13 group00390, chr13 group00391, chr13 group00395,chr13 group00399 and PROZ for trisomy 13 (U.S. Pat. No. 8,476,013); CpGhypermethylation sites at 11p15.5 for Beckwith-Wiedemann Syndrome(Coffee et al., Genet Med, 2006 8:628); 15q11.2-q13 for Prader-Willi andAngelman syndromes (Procter et al., Clin Chem, 2006 52:1276); the XLlocus associated with Albright's Hereditary Osteodystrophy (Izzi et al.,PLoS ONE, 2012 7:e38579); and CpG sites in the FMR1 gene for Fragile XSyndrome (Hansen et al., Hum Mol Genet, 1992 1:571; Alisch et al., BMCMed Genet, 2013 14:18). Thus, a splint oligonucleotide of the subjectmethod may be designed to be complementary to the hypermethylated sitesof these loci to diagnose prenatal genetic and epigenetic anomalies.

In certain embodiments, the present method may be used for NIPT. Forinstance, hypermethylated sequences in the promoter region of theRASSF1A gene may be used to improve prenatal RhD genotyping anddetection of susceptibility to preeclampsia (Chan et al., Clin Chem,2006 52:2211; Tsui et al., Prenat Diagn, 2007 27:1212). As such, asplint oligonucleotide of the subject method may be designed to becomplementary to the methylated sequences in the promoter region of theRASSF1A gene. RASSF1 (Ras association (RalGDS/AF-6) domain family 1) wasfound to be hypermethylated in placenta but completely unmethylated inmaternal blood cells.

In certain embodiments, the present method may be used to detecthypermethylation in fetal DNA by designing a splint oligonucleotidecomplementary to methylated sequences in or associated with thefollowing genes: SOX14, TBX3, SIX2, TLX3, FOXP4, NPY, SHH, OSR2, GLIS3,PRMT8, PAX9, SIX1, ISL2, DLX4, CBX4 and EDG6 (Nygren et al., Clin Chme,2010, 56: 1627; U.S. Pat. No. 8,476,013).

In yet other embodiments, the present method may be used to diagnosesusceptibility to pathological pregnancies, such as preeclampsia. Forexample, hypermethylation in CpG sites in the c-myc promoter region andexon 1 of H19 of fetal DNA are associated with preeclampsia (Rahat etal., Mol Hum Reprod, 2014; Lu et al., Int J Mol Med, 2014). Thus incertain embodiments, a splint oligonucleotide of the subject methoddesigned to be complementary to the methylated sequences in theseregions may be used for determining predisposition to preeclampsia.

In certain embodiments, the subject method may be employed as part of acancer diagnostic. For example, colorectal cancer is associated withincreased methylation of CpG islands in or around several genes,including BMP3 NDRG4, Septin 9, TFPI2, p14, EYA2, ALX4, IGFBP7, GATA4/5,MGMT, TBX5, ID4, BTG4, miRNA-34b/c, CDH13, TPEF/HPP1, NPY, PENK, WIF,EN1, SCTR, INHBB and Vimentin (Zou et al., Cancer Epidemiol BiomarkersPrev, 2007 16:2686; Melotte et al., J Natl Cancer Inst, 2009 101:916;Grutzmann et al., PLoS One, 2008 3:e3759; DeVos et al., Clin Chem, 2009,55:1337; Wasserkort et al., BMC Cancer, 2013 13:398; Hibi et al., CancerLett, 2011 311:96; Esteller et al., Cancer Res, 2000 60:129; Zou et al.,Cancer Epidemiol Biomarkers Prev, 2007 16:2686; Hinoue et al., PLoS One2009 4:e8357; Hellebrekers et al., Clin Cancer Res, 2009 15:3990; Shenet al., J Natl Cancer Inst, 2005 97:1330; Yu et al., Oncogene, 201029:6464; Clin Cancer res, 2004 10:7475; Toyota et al., Cancer Res, 200868:4123; Toyooka et al., Cancer Res, 2002 62:3382; Ebert et al.,Neoplasia, 2005, 7:771; Chen et al., J Natl Cancer Inst, 2005 97:112,Cancer Epidemiol Biomarkers Prev, 2007 16:2686; Roperch et al., BMCCancer, 2013 13:566; Mayor et al., Br J Cancer, 2009 100:1534). As such,in certain embodiments, the splint oligonucleotide used in the subjectmethod may be designed to be complementary to a methylated sequence inthose genes.

Hepatocellular carcinoma and/or liver cirrhosis and/or hepatitis C virusinfection is correlated with increased methylation of CpG islandsassociated with p16, p15, RASSF1A, SSBP2, B4GALT1, CASP8, SOCS1, theD17S5 locus of HIC-1, APC, WIF-1, RUNX-3, DLC-1, SFRP-1, DKK, CDH1,KLK10, OCGR1, DUSP4, NPR1, CYP24A1, CDKN2A, CCNA1, GSTP1, p14, p73,RAR-β, AR, DBCCR1, IRF7 and OCT6 (Wong et al., Cancer Res, 1999 59:71;Chu et al., J Korean Med Sci, 2004 19:83; Wong et al., Clin Cancer Res,2000 6:3516; Michailidi et al., Gastroenterol Res Pract, 20142014:597164; Yu et al., BMC Cancer, 2002 2:29; Yoshikawa et al., NatGenet, 2001 28:29; Kanai et al., Hepatology, 1999 29:703; Liu et al.,World J Gastroenterol, 2011 17:4718; Mah et al., Biomark Res, 2014 2:5).As such, in certain embodiments, the splint oligonucleotide used in thesubject method may be designed to be complementary to a methylatedsequence in these regions.

Likewise, multiple myeloma is associated with increased methylation ofCpG islands in the promoter regions of p16 and SOCS-1 (Merlo et al., NatMed, 1995 1:686; Lo et al., Cancer Res, 1999 59:3899; Wong et al., ClinCancer Res, 2003 9:1047; Galm et al., Blood, 2003, 101:2784). As such,in certain embodiments, the splint oligonucleotide used in the subjectmethod may be designed to be complementary to a methylated promotersequence in those genes. In addition, multiple forms of leukemia areassociated with hypermethylation in p15, p16, p14, p53, DAPK, NES-1,ADAMTS5, WIF-1, sFRP-1, MYOD1, PTPRZ1, PPARG, FOXE3, FBXO39, PKDREJ,TCF3, EGR4, BTG4, PAX5, IKZF1, TLX3, RAG1, POU2AF1, COBL, COL6A2, CPVL,DFNB31, EYA4, FAM24B, FAT1, FUCA2, INADL, MYO3A, PCDHGA12, PON3, ROR1,SYNM, TNIK, ZNF502, CDKN2A, PTPRO, CSMD1, ABI3, SCGB2A1, VHL, GPX3,IGSF4, SERPINDS, ADORA3, AIRE, CARD15, LOC340061, UNCSCL, LDOC1, PRF1,FABP7, SOX11, DLX1, FAM62C, SOX14, RSPO1, ADCYS, HAND2, SPOCK, MLL,ING1, PRIMA1, BCL11B, LTBP2, BNC1, NR2F2, SALL1, GALGT2, LHX1, DLX4,KLK10, TFAP2, APP, F1121062, BNIP3, MGMT, RBP1, GATA4, CRABP1, LANCL1,KCNK12, SORL1, CXorf57, SOX9, KIAA0746, ASPHD2, ARHGAP17, PMM2, IL12A,JDP2, PAK1, GALNS, FGD2, LYAR, HOXA9, AHR, ROBO1, NPTX2, CDH1, CDKN2B,HOXD8, MLF-1, PCDH8, CD44, GADD45, ZMAT3, IRF7, KLF6 and p73 (Bodoor etal., Asian Pac J Cancer Prev, 2014 15:75; Zhao et al., Biomark Res, 20131:24; Martinez-Delgado et al., Int J Cancer, 2002 102:15). Thus, incertain embodiments, the splint oligonucleotide used in the subjectmethod may be designed to be complementary to a methylated sequence inthose genes.

Likewise, cholangiocarcinoma is associated with hypermethylation of CpGislands associated with OPCML, SFRP1, HIC1, PTEN and DcR1 (Sriraksa etal., Br J Cancer, 2011 104:1313). As such, in certain embodiments, thesplint oligonucleotide used in the subject method may be designed to becomplementary to the methylated CpG islands of these genes.

Furthermore, lung cancer is associated with increased methylation of CpGislands in the promoter regions of p16, DAP kinase, MGMT, SRBC, GSTP1(Belinski et al., Proc Natl Acad Sci, 1998 95:11891; Esteller et al.,Cancer Res, 1999 59:67; Esteller et al., Cancer Res, 1999 59:67;Esteller et al., Proc Am Assoc Cancer Res, 1998 39:92; Zöchbauer-Mülleret al., Oncogene, 2005 24:6249; Esteller et al., Cancer Res, 1999 59:67;Jain et al., PLoS One, 2012 7:e35789). As such, in certain embodiments,the splint oligonucleotide used in the subject method may be designed tobe complementary to a methylated sequence associated with these loci.

In addition, breast cancer is associated with hypermethylation of CpGislands in SFRP1, SFRP2, SFRP 5, BRCA1, LKB1, ER, PR, SYK, RIZ1, GSTP1(Veeck et al., Oncogene, 2006 25:3479; Veeck et al., Mol Cancer, 20087:83; Veeck et al., Carcinogenesis, 2008 29:991; Esteller et al., J NatlCancer Inst, 2000 92:564; Esteller et al., Cancer Res, 2001 61:3225;Esteller et al., Oncogene, 2000 19:164; Ottaviano et al., Cancer Res,1994 54:2552; Lapidus et al., Clin Cancer Res, 1996 2:805; Yuan et al.,Cancer Res, 2001 61:5558; Du et al., Cancer Res, 2001 61:8094; Estelleret al., Cancer Res, 1998 58:4515). As such, in certain embodiments, thesplint oligonucleotide used in the subject method may be designed to becomplementary to a methylated sequence in or to a methylated sequenceassociated with these genes.

Likewise, renal carcinoma is associated with increased methylation ofCpG islands in the promoter regions of GSTP1 and VHL (Esteller et al.,Cancer Res, 1998 58:4515; Herman et al., Proc Natl Acad Sci, 199491:9700); endometrial carcinoma is associated with increased methylationin the promoter regions of hMLH1 (Esteller et al., Am J Pathol, 1999155:1767); and esophageal adenocarcinoma is associated withhypermethylation in the promoter regions of APC (Kawakami et al., J NatlCancer Inst, 2000 92:1805). Thus, in certain embodiments, the splintoligonucleotide used in the subject method may be designed to becomplementary to a methylated sequence in or to a methylated sequence inthe promoter regions of these genes.

Furthermore, oral squamous cell carcinoma is associated withhypermethylation in KIF1A, HOXA9, NID2, EDNRB, p16, RARβ, CDH-1, CYGBand CYCA1 (Guerrero-Preston et al., Cancer Prev Res (Phila), 20114:1061; Shaw et al., Br J Cancer, 2006 94:561); while hypermethylationin EDNRB and DCC are associated with premalignant or malignant orallesions (Pattani et al., Cancer Prev Res (Phila), 2010 3:1093; Schusselet al., Clin Cancer Res, 2013 19:3268). As such, in certain embodiments,the splint oligonucleotide used in the subject method may be designed tobe complementary to a methylated sequence in the promoter regions ofthese genes. In addition, esophageal squamous cell carcinoma isassociated with increased methylation of 5′ regulatory regions ofmiRNAs, including miR-34a, miR-34b/c and miR-129-2 (Chen et al., Int JCancer, 2012 130:1607). Thus, in certain aspects, the splintoligonucleotide used in the subject method may be designed to becomplementary to a methylated sequence in the 5′ regulatory regions ofthese miRNAs. Esophageal squamous cell carcinoma is also associated withincreased methylation in GPX3 (He et al., Dig Dis Sci, 2011 56:681); andECRG4 (Yue et al., World J Gastroenterol, 2003 9:1174). As such, incertain embodiments, the splint oligonucleotide used in the subjectmethod may be designed to be complementary to a methylated sequence inthe promoter regions of these genes.

Likewise, vulvar squamous cell carcinoma is associated withhypermethylation in MGMT, RASSF2A and TSP-1 (Guerrero et al., Int JCancer, 2011 128:2853). As such, in certain embodiments, the splintoligonucleotide used in the subject method may be designed to becomplementary to a methylated sequence in the promoter regions of thesegenes. Epstein-Barr virus-associated gastric carcinomas is associatedwith hypermethylation at loci such as MINT2, MINT31, p14, p16, p′73, andRUNX3 (Saito et al., J Med Virol, 2013 85:121). Thus the splintoligonucleotide used in the subject method may be designed to becomplementary to a methylated sequence at those loci.

In addition, prostate cancer is associated with hypermethylation inGSTP1, AR, TIMP2 (Lee et al., Proc Natl Acad Sci, 1994 85:11733; Jarrardet al., Cancer Res, 1998 58:5310; Pulukuri et al., Oncogene, 200726:5229). As such, in certain embodiments, the splint oligonucleotideused in the subject method may be designed to be complementary to amethylated sequence in these genes. Retinoblastoma is associated withhypermethylated promoter sequences of genes, such as Rb (Stirzaker etal., Cancer Res, 1997 57:2229).

Therefore, the splint oligonucleotide used in the subject method may bedesigned to be complementary to a methylated sequence in the promoterregions of these genes. Likewise, glioblastoma multiforme is associatedwith hypermethylation of CpG sites in the 5′ regulatory region of THBS1(Li et al., Oncogene, 1999 18:3284). In certain embodiments, the splintoligonucleotide used in the subject method may be designed to becomplementary to a methylated sequence in these genes.

In some embodiments, the subject method may be employed to diagnoseneurodegenerative disorders. For instance, hypermethylation of CpG sitesat C9orf72 is associated with amyotrophic lateral sclerosis andfrontotemporal lobar degeneration (Xi et al., Am J Hum Genent, 201392:981; Xi et al., Am J Hum Genent, 2014). Thus in certain embodiments,a splint oligonucleotide of the subject method may be designed to becomplementary to a methylated sequence in CpG sites of C9orf72.Likewise, increased methylation at CpG sites at the following genes areassociated with Parkinson's Disease: KCTD5, VAV2, MOG, TRIM10, HLA-DQA1,ARHGEF10, GFPT2, HLA-DRB5, TMEM9, MRI1, MAPT, HLA-DRB6, LASS3, GSTTP2,GSTTP1 (Masliah et al., Epigenetics 2013 8:1030; Coupland et al., MovDiord, 2013). As such, a splint oligonucleotide of the subject methodmay be designed to be complementary to a methylated sequence in the CpGsites of those genes.

As noted above, in some cases the sample analyzed may be a sample ofcell-free DNA obtained from blood, e.g., from the blood of a pregnantfemale. In these embodiments, the method may be used to detectchromosome abnormalities in the developing fetus (as described above) orto calculate the fraction of fetal DNA in the sample, for example. Theseembodiments provide for the detection and quantification of fetalnucleic acid in a maternal sample based on the methylation status of thenucleic acid in the sample. In some cases, the amount of fetal nucleicacid from a maternal sample can be determined relative to the totalamount of nucleic acid present, thereby providing the percentage offetal nucleic acid in the sample. In some cases, the copy number offetal nucleic acid can be determined in a maternal sample. In somecases, the amount of fetal nucleic acid can be determined in alocus-specific manner and sometimes with sufficient sensitivity to allowfor accurate chromosomal dosage analysis (for example, to detect thepresence or absence of a fetal aneuploidy). In some cases, the methodcan be used to determine the concentration of fetal DNA in a maternalsample, for example, by the following method: a) determining the totalamount of DNA present in a maternal sample; determining the amount of amethylated fetal marker in the material sample using the present method;and comparing the amount of total DNA to the amount of the fetal makerDNA. The concentration of fetal DNA in the maternal sample can beextrapolated from these results. In some cases, the absolute copy numberof fetal nucleic acid in a maternal sample can be determined.

In particular embodiments, a method of estimating the fetal fraction ina sample of cell-free DNA obtained from the blood of a pregnant femaleis provided. In some cases, this method comprises; (a) digesting saidsample with an MspJI family restriction endonuclease to produce apopulation of fragments that are in the range of 20-40 base pairs inlength and have a central methylated cytosine; (b) ligating adaptorsequence A and adaptor sequence B to the respective ends of a pluralityof target fragments that are only methylated in fetal DNA, by: (i)hybridizing a plurality of splint oligonucleotide of formula B′-X′-A′ tothe fragments of (a) in the presence of the adaptor sequences A and B,wherein A′ and B′ are complementary to A and B, respectively, andsequence X′ varies between the different splint oligonucleotides andeach sequence X′ is complementary to a target fragment that is onlymethylated in fetal DNA; and (ii) ligating adaptor sequence A, sequenceX and adaptor sequence B to one another to produce products of formulaA-X-B; and (c) quantifying the amount of products of formula A-X-B, and(d) normalizing the amount obtained in (c) to the amount of controllocus in the sample, wherein said normalizing provides an estimate ofthe fetal fraction in the sample. In some cases, this method may beimplemented using at least 100, at least 200, at least 300, at least 500or at least 1,000 or more different splint oligonucleotides, where eachsplint oligonucleotide is complementary to a sequence that is onlymethylated in fetal DNA. In these cases, the fetal fraction canestimated by, e.g., comparing the normalized amount obtained in (d) to astandard curve. In some cases, the control locus may be on one or moreof chromosomes 21, 18 and 13. In some cases, the amount of ligationproduct quantified in (c) may be compared to the amount of a controllocus that is not differentially methylated in the sample.

In certain embodiments, the double stranded DNA being analyzed may bederived from a single source (e.g., a single subject, etc.), whereas inother embodiments, the nucleic acid sample may be a pool of nucleicacids extracted from a plurality of sources (e.g., a pool of nucleicacids from a plurality of subjects, etc.), where by “plurality” is meanttwo or more. As such, in certain embodiments, a nucleic acid sample cancontain nucleic acids from 2 or more sources, 3 or more sources, 5 ormore sources, 10 or more sources, 50 or more sources, 100 or moresources, 500 or more sources, 1000 or more sources, 5000 or moresources, up to and including about 10,000 or more sources. Molecularbarcodes may allow the sequences from different sources to bedistinguished after they are analyzed.

Kits

Also provided by this disclosure are kits for practicing the subjectmethods, as described above. In some embodiments, a kit may contain atleast: (a) an MspJI family restriction endonuclease; (b) adaptorsequence A and adaptor sequence B; (c) a splint oligonucleotide offormula B′-X′-A′, wherein X′, A′ and B′ are complementary to X, A and B,respectively, and X is a genomic sequence of 20-40 base pairs in lengthand has a central cytosine that is differentially methylated; and (d) aligase. In some embodiments, the adaptor sequences A and B may bepresent in the same oligonucleotide molecule. The various components ofthe kit may be present in separate containers or certain compatiblecomponents may be precombined into a single container, as desired.

In addition to the above-mentioned components, the subject kits mayfurther include instructions for using the components of the kit topractice the subject methods, i.e., instructions for sample analysis.The instructions for practicing the subject methods are generallyrecorded on a suitable recording medium. For example, the instructionsmay be printed on a substrate, such as paper or plastic, etc. As such,the instructions may be present in the kits as a package insert, in thelabeling of the container of the kit or components thereof (i.e.,associated with the packaging or subpackaging) etc. In otherembodiments, the instructions are present as an electronic storage datafile present on a suitable computer readable storage medium, e.g.,CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g., via the internet, are provided. An exampleof this embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

1-18. (canceled)
 19. A kit comprising: (a) an MspJI family member; (b)adaptor sequence A and adaptor sequence B, (c) a splint oligonucleotideof formula B′-X′-A′, wherein X′, A′ and B′ are complementary to X, A andB, respectively, and X is a genomic sequence of 20-40 base pairs inlength and has a central cytosine that is differentially methylated; and(d) a ligase.
 20. The kit of claim 19, wherein adaptor sequences A and Bare present in same oligonucleotide molecule.
 21. The kit of claim 19,wherein the MspJI family restriction endonuclease is MspJI.
 22. The kitof claim 19, wherein the genomic sequence is a sequence from the humangenome.
 23. The kit of claim 19, wherein sequences A, B, A′ and B′ arein the range of 15 to 100 bases nucleotides in length.
 24. The kit ofclaim 22, wherein sequences A, B, A′ and B′ are not in the human genome.25. The kit of claim 19, wherein the central cytosine is methylated incancer cells but not in normal cells.
 26. The kit of claim 19, whereinthe central cytosine is methylated in fetal DNA but not in maternal DNA.